DESCRIPTION: The long-term goal of this project is an understanding of structure-function relationships in the Na,K-pump. The investigator has proposed studies in protein chemistry and affinity labeling that are a straightforward extension of his previous work. The cation complexation site within the catalytic alpha subunit of the pump will be identified by affinity labeling with positively charged carboxyl-reactive compounds (Specific Aim 1). The isothiocyanate-reactive lysine in the major cytoplasmic domain (Lys-501) will be used as an anchor in cross-linking experiments designed to identify those residues involved with nucleotide binding and energy transduction (Aim 2). Additional cross-linking and photolabeling will be used in right-side-out microsomes to test directly the proposed membrane topology derived from hydropathy analysis (Aim 3). Finally, photoactive derivatives of the cardiotonic steroids will be used to identify amino acids that contribute to glycoside binding (Aim 4).